Detection of the presence of a specific DNA or RNA sequence in a sample is required for a variety of experimental, diagnostic and therapeutic purposes, e.g. detection of a specific mutation in a sample of amniotic fluid, parenterage testing, testing for incorporation of a viral DNA into a cell's genomic DNA, etc. The task of direct detection of a specific DNA or RNA sequence, which is routinely performed by the use of an appropriately labelled probe, is often hindered by the fact that the specific DNA or RNA is present in a sample only in minute amounts.
Examples of methods which enable the amplification of DNA sequences present in a sample in only minute quantities are: LCR (ligase chain reaction), 3SR (self-sustained sequence replication) or PCR (polymerase chain-reaction). In PCR a sample is contacted with a primer DNA complimentary to a 3' end sequence of the specific DNA, a DNA polymerase and with single DNA nucleotides. Following a number of replication cycles, the sample is enriched with the specific assayed DNA. A typical cycle of PCR comprises three distinct stages: a first stage in which the double-stranded DNA is melted to two single strands; a second stage of annealing of the primer to the single-stranded DNA; and a third stage of polymerization where the annealed primers are extended by the DNA polymerase, to produce a double-stranded DNA. The cycle of melting, annealing and DNA synthesis is repeated many times, the products of one cycle serving as templates for the next ad thus, each successive cycle enriches the sample with the specific DNA.
PCR suffers from several shortcomings, the most serious of which being its lack of specificity. The effective hybridization temperature, i.e. the temperature in which the two strands of DNA hybridize, determines the specificity of the reaction. A low effective hybridization temperature results in a higher percentage of non-specific binding. In PCR this temperature, which is defined by the temperature of the annealing stage, is relatively low and this brings about non-specific binding of the probe to the target sequences resulting in amplification of undesired sequences which brings about a relatively high background reading.
This non-specificity also requires an additional and time-consuming detection procedure such as electrophoretic separation of the amplification products on an agarose gel, in order to separate between the various amplification products, and does not enable detection of the presence of the assayed DNA by a mere detection of amplification.
PCR also suffers from a severe problem of contamination which is due to amplification of sequences that did not originate from the test sample being sequences unintentionally introduced to the sample.
Another disadvantage of PCR is that it is a complex procedure. Typically, each of the stages of melting, annealing and polymerization is carried out at a different temperature, e.g. melting at 94.degree. C., annealing at 50.degree. C. and polymerization at 72.degree. C. Since the samples have to be constantly cycled through several temperatures a special apparatus is required rendering the procedure laborious and time consuming.
Another shortcoming of PCR is in the time required therefor. A typical cycle lasts several minutes, and usually 25-30 cycles are required to produce sufficient copies of amplified DNA. Thus, a typical PCR even in a completely automated system lasts at least 2 to 3 hours.
Finally, PCR is basically suited for the detection of DNA sequences. Where detection of RNA sequences is desired, RNA has to be converted first to DNA (by reverse transcription). This conversion to DNA requires additional time, effort and enzymes, and also introduces many errors due to the inherent inaccuracy of reverse transcription.
It should be noted that although PCR is advantageous in obtaining large amounts of a specific DNA, such as for producing large quantities of probes for genetic assays, it is often an "over-kill" where merely the presence of a specific DNA sequence in a sample is to be assayed.
Other such methods such as 3SR (WO PCT 89/05631) and Target Nucleic Acid Amplification/Detection (WO PCT 89/05533) are relatively rapid isothermal processes for DNA detection. However, these methods also suffer from relatively effective low hybridization temperatures which are even lower than those of PCR, typically in the range of 37-41.degree. C. These low temperatures drastically reduce the specificity of the procedure due to non-specific probe-target binding, and in cases of clinical diagnostics, this may result in an intolerable level of misdiagnosis.
Additionally, amplification strategies such as Target Nucleic Acid Amplification/Detection that are based on the amplification properties of a replicase-type enzyme are unreliable due to the possibility of spontaneous RNA amplification in the absence of target (Chetverin-AB, et al., J. Mol. Biol., 222(1), 3-9 (1991)).
It is the object of the invention to provide a method for the detection of a nucleic acid sequence which is:
(i) reliable and sequence specific due to the minimalization of incorrect target-probe hybridization;
(ii) relatively rapid;
(iii) essentially isothermic eliminating the need for specialized and expensive apparatus;
(iv) relatively simple, not requiring the addition of a large number of different enzymes or nucleotide pools; and
(v) amenable to automation by enabling the amplification process itself to be indicative of the presence or absence of the nucleic acid sequence to be assayed.
U.S. Pat. No. 5,434,047 teaches a method for ensuring that only hybrids which are perfectly matched between a probe sequence (termed "target probe") and a nucleic acid sequence present in a sample (termed "target nucleotide sequence") are formed, while imperfect matches between the probe and other sequences present in the assayed sample (termed "non-target nucleotides") are not formed. The method involves adding to the reaction mixture blocker molecules which are complementary to the non-target nucleotides which are present in the assayed sample. These blocker molecules, hybridize with the non-target nucleotide in the assayed sample, avoiding their hybridization with the target probe, and thus eliminate production of false-positive results. Each blocker molecule, of U.S. Pat. No. 5,434,047, is specific only to one type of non-target nucleotide, and is emphatically not universal in all assay kits. For example, where it is desired to assay a sample for the presence of a specific nucleic acid sequence ("target nucleotide") which is indicative of a specific bacteria species, a battery of different blocker molecules, each complementary to a nucleic acid sequence of other species of bacteria ("non-target nucleotides") have to be constructed. If, for some reason, not all possible non-target nucleotide combinations were predicted, and consequently not all types of complementary blocker molecules were constructed, the blocker molecule would not avoid imperfect matches with the labeled probe, thus providing a false-positive result.
It would have been desirable to construct a universal single blocker molecule, which would be suitable for elimination of all imperfect hybridizations between a probe and nucleic acid sequences present in a sample, and thus eliminate all positive results, even in the presence of many types of non-target nucleotide sequences.
Further objects of the invention will become clear from the following description.
Glossary
Below are the meanings of some of the terms which will be used in the following description and claims. For ease of reference, the reader is also referred to the accompanying drawings (the numbers in brackets in the Glossary below refer to the item numbers in the drawings):
Assayed nucleic acid sequence (102,202,302,402,502,602,1402)--The DNA or RNA sequence which presence in the sample is to be detected. PA0 First DNA molecule (220,320,420,520,620,1420)--a DNA molecule having a double-stranded, i.e. functional promoter and a 5' end sequence which is complementary to the 5' end portion of the assayed nucleic acid sequence (102, . . . etc.). PA0 Second DNA molecule (222,322,432,522,622,1422)--a DNA molecule comprising a single-stranded 3' end sequence being complementary to the 3' end portion of the assayed nucleic acid sequence (102, . . . etc.) and further comprising a sequence which can be transcribed to the triggering RNA sequence (see below). The 3' end sequence of the second DNA molecule and the 5' end sequence of the first DNA molecule may be complementary to the entire assayed nucleic acid sequence or to only a part thereof, leaving an intermediary portion in the assayed nucleic acid sequence having no complementary counterparts in either the first or second DNA molecules. PA0 Third DNA molecule (623)--a single-stranded DNA molecule complementary to the intermediary portion of the assayed nucleic acid sequence. PA0 Detection ensemble (104,204)--an ensemble of molecules comprising the first DNA molecule (220, . . . etc.), the second DNA molecule (222, . . . etc.), and where the 5' end sequence and the 3' end sequence of the first and second DNA molecule, respectively, are complementary together to only a portion of the assayed nucleic acid sequence also comprising the third DNA molecule (623). The detection ensemble optionally comprises also a ligase. In the presence of the assayed DNA (102, . . . etc.) and the transcription reagents (see below) the detection ensemble is activated and an RNA transcript (110,210,310,410,510,610,710,810,910,1010,1110,1210,1310,1410,1510, 1610) comprising the triggering RNA sequence (see below) is produced. PA0 Triggering RNA sequence--a sequence in the RNA transcript (110, . . . etc.) transcribed from the second DNA molecule (222, . . . et.), which is only produced after activation of the detection ensemble. This RNA sequence is then capable of triggering transcription in the RNA amplification ensemble (see below) of a signal RNA molecule (see below) comprising the signal RNA sequence (see below). PA0 Triggering RNA molecule (110,210,310,410,510,610,710,810,910,1010, 1110,1210,1310,1410,1510,1610)--the RNA molecule comprising the triggering RNA sequence. PA0 Signal RNA sequence--a sequence in the transcription product of the RNA amplification ensemble (see below). The production of the signal RNA sequence, above a baseline level, indicates the presence of the assayed nucleic acid sequence in the sample. PA0 Signal RNA molecule (116,716,816,916,1016,1116,1216)--the RNA molecule comprising the signal RNA sequence. PA0 Signal DNA sequence--a DNA sequence serving as a template from which the signal RNA sequence is transcribed. PA0 Transcription reagents (113,213,413,513,713,813,913,1013,1113,1213, 1313,1413,1513,1613)--RNA polymerase with single RNA nucleotides and buffers required for RNA transcription. PA0 Transcription system--a DNA homoduplex or a DNA/RNA heteroduplex comprising a functional promoter and a downstream DNA or RNA sequence which can be transcribed upon activation of the promoter into an RNA transcript. PA0 RNA amplification ensemble (FIGS. 7-13, 15,16)--an ensemble comprising essentially the triggering RNA molecule (110, . . . etc.) and the transcription reagents (213, . . . etc.) in the first embodiment of the invention; or the triggering RNA molecule (116, . . . etc.), the transcription reagents (213, . . . etc.) and fourth DNA molecule (see below) in the second embodiment; or the triggering RNA, the transcription reagents and a fifth and sixth DNA molecules (see below), in the third embodiment of the invention; or the triggering RNA, the transcription reagents, and a seventh and eighth DNA molecule in the fourth embodiment of the invention. The RNA amplification ensemble optionally comprises a ligase. In one embodiment of the first embodiment, the presence of the triggering RNA together with the transcription system is sufficient for the transcription of the signal RNA sequence. In the second embodiment, the triggering RNA hybridizes with the fourth DNA molecule. In the third embodiment, the triggering RNA hybridizes with the fifth and sixth DNA molecules bringing them together. In the fourth embodiment the triggering RNA hybridizes with the seventh or eighth DNA molecules. The RNA/DNA hybrid produced in accordance with the second, third and fourth embodiments, serves as a template for the production of the signal RNA sequence. PA0 Fourth DNA molecule--a DNA molecule (1148) which is part of the RNA amplification ensemble in accordance with the second embodiment. This molecule comprises a promoter which is single-stranded in at least an essential part thereof and is thus inactive. It further comprises the signal DNA sequence. When the triggering RNA sequence, which in this embodiment is complementary to the single-stranded part of the promoter, hybridizes with the single-stranded part of the promoter of the fourth DNA molecule, a functional promoter is produced and thus the signal RNA molecule can be transcribed. PA0 Fifth DNA molecule--a molecule (1252) which is part of the amplification ensemble in accordance with the third embodiment. It comprises a functional promoter, and at its 5' end, a single-stranded sequence which is complementary to the 5' end portion of the triggering RNA sequence. PA0 Sixth DNA molecule--a molecule (1259) which is part of the amplification ensemble in accordance with the third embodiment. It comprises at its 3' end a single-stranded sequence which is complementary to the remaining 3' end portion of the triggering RNA sequence and in addition, comprises the signal DNA sequence. PA0 Seventh DNA molecule--a molecule (1580) which is part of the amplification ensemble in accordance with the fourth embodiment. It has a functional, double-stranded promoter, either a priori prepared or assembled from two single stranded sequences, linked to an antisense sequence complementary to the 3' end sequence of the triggering RNA. One or a few end nucleotides in the 5' end of the template strand of this molecule could be RNA nucleotides. The 3' end sequence of the triggering RNA hybridizes to said antisense sequence and after ligation the promoter can induce RNA transcription, the triggering RNA serving as a template. PA0 Eighth DNA molecule--a molecule (1529') which is part of the amplification ensemble in accordance with the fourth embodiment. It is similar to the seventh DNA molecule, the difference being in the antisense sequence which in the eighth DNA molecule is identical to the 5' end sequence of the triggering RNA molecule. The transcription product of the seventh DNA molecule/triggering RNA hybrid can thus hybridize to the eighth molecule and the so formed hybrid serves there as a template for transcription of RNA molecule having the sequence of the triggering RNA sequence, which in turn can activate again the seventh DNA molecule in a "ping-pong" manner. PA0 Promoter molecule--a DNA molecule (1429) which is part of the detection ensemble according to the fourth embodiment of the invention and is essentially identical to the eighth DNA molecule. PA0 Adapter molecule--a DNA molecule (1431) comprising a sequence complementary to the non-template sequence immediately adjacent the promoter sequence in the promoter molecule possibly having one or a few RNA nucleotides at its 3' end. PA0 Joiner molecule--a DNA molecule (1433) comprising at its 5' end a sequence which is complementary to a 5' end portion of the adapter molecule and a sequence in the remaining 3' portion of the molecule which is complementary to 3 end portion of the first molecule in accordance with the fourth embodiment. The joiner molecule serves for joining the first and the adapter molecule in the fourth embodiment. PA0 DNA iniation sequence (DIS)--a DNA sequence present downstream of the promoter of the seventh or eighth DNA molecules which enhances transcription of the sequence present downstream therefrom, by the RNA polymerase. PA0 Probe nucleic acid sequence--a nucleic acid sequence which is complementary to a pre-defined nucleic acid sequence whose presence is to be detected in the assayed sample. PA0 Blocker nucleic acid sequence--a nucleic acid sequence which is complementary to the probe nucleic acid sequence. PA0 Perfectly matched--two nucleic acid sequences which are fully complementary to one another. PA0 Non-perfectly matched--two nucleic acid sequences which are not fully complementary to one another. PA0 (a) reacting the sample with a detection ensemble comprising: PA0 (b) incubating under conditions to allow hybridization of said first DNA molecule and said second DNA molecule and were present also said third DNA molecule with said assayed nucleic acid sequence, and optionally adding a ligase to allow ligation of adjacent ends of said first, second and third DNA molecules; PA0 (c) adding transcription reagents comprising an RNA polymerase and RNA nucleotides and incubating under conditions to allow the formation of RNA transcripts having said triggering RNA sequence; PA0 (d) contacting the RNA transcripts with an RNA amplification ensemble in which the triggering RNA sequence induces formation of RNA molecules containing the signal RNA sequence; and PA0 (e) detecting the presence of said signal RNA sequence, positive results indicating the presence of said assayed nucleic acid sequence in said sample. PA0 (a) incubating the sample and the probe nucleic acid sequence under conditions allowing hybridization of matched nucleic acid sequences; PA0 (b) increasing the temperature of the reaction mixture to such which is below the melting point of perfectly matched hybridized nucleic acid sequences but above that which leads to melting of non-perfectly matched hybridized nucleic acid sequences; PA0 (c) adding an amount of a blocker nucleic acid sequence, having a sequence which perfectly matches the sequence of said probe nucleic acid sequence or matches an essential part of said probe, the blocking sequence being sufficiently long to block hybridization of the nucleic acid sequence contained in the sample to probe nucleic acid sequence upon lowering of temperature; PA0 (1) hybrids between probes and sequences in the assayed sample which are perfectly matched; PA0 (2) hybrids between probes and sequences in the assayed sample which are non-perfectly matched.